The present invention relates to compounds which elevate pyruvate dehydrogenase (PDH) activity, processes for their preparation, pharmaceutical compositions containing them as active ingredient, methods for the treatment of disease states associated with reduced PDH activity, to their use as medicaments and to their use in the manufacture of medicaments for use in the elevation of PDH activity in warm-blooded animals such as humans.
Within tissues adenosine triphosphate (ATP) provides the energy for synthesis of complex molecules and, in muscle, for contraction. ATP is generated from the breakdown of energy-rich substrates such as glucose or long chain free fatty acids. In oxidative tissues such as muscle the majority of the ATP is generated from acetyl CoA which enters the citric acid cycle, thus the supply of acetyl CoA is a critical determinant of ATP production in oxidative tissues. Acetyl CoA is produced either by xcex2-oxidation of fatty acids or as a result of glucose metabolism by the glycolytic pathway. The key regulatory enzyme in controlling the rate of acetyl CoA formation from glucose is PDH which catalyses the oxidation of pyruvate to acetyl CoA and carbon dioxide with concomitant reduction of nicotinamide adenine dinucleotide (NAD) to NADH.
In disease states such as both non-insulin dependent (NIDDM) and insulin-dependent diabetes mellitus (IDDM), oxidation of lipids is increased with a concomitant reduction in utilisation of glucose, which contributes to the hyperglycaemia. Reduced glucose utilisation in both IDDM and NIDDM is associated with a reduction in PDH activity. In addition, a further consequence of reduced PDH activity may be that an increase in pyruvate concentration results in increased availability of lactate as a substrate for hepatic gluconeogenesis. It is reasonable to expect that increasing the activity of PDH could increase the rate of glucose oxidation and hence overall glucose utilisation, in addition to reducing hepatic glucose output. Another factor contributing to diabetes mellitus is impaired insulin secretion, which has been shown to be associated with reduced PDH activity in pancreatic xcex2-cells (in a rodent genetic model of diabetes mellitus Zhou et al. (1996) Diabetes 45: 580-586).
Oxidation of glucose is capable of yielding more molecules of ATP per mole of oxygen than is oxidation of fatty acids. In conditions where energy demand may exceed energy supply, such as myocardial ischaemia, intermittent claudication, cerebral ischaemia and reperfusion, (Zaidan et al., 1998; J. Neurochem. 70: 233-241), shifting the balance of substrate utilisation in favour of glucose metabolism by elevating PDH activity may be expected to improve the ability to maintain ATP levels and hence function.
An agent which is capable of elevating PDH activity may also be expected to be of benefit in treating conditions where an excess of circulating lactic acid is manifest such as in certain cases of sepsis.
The agent dichloroacetic acid (DCA) which increases the activity of PDH after acute administration in animals, (Vary et al., 1988; Circ. Shock, 24: 3-18), has been shown to have the predicted effects in reducing glycaemia, (Stacpoole et al., 1978; N. Engl. J. Med. 298: 526-530), and as a therapy for myocardial ischaemia (Bersin and Stacpoole 1997; American Heart Journal, 134: 841-855) and lactic acidaemia, (Stacpoole et al., 1983; N. Engl. J. Med. 309: 390-396).
PDH is an intramitochondrial multienzyme complex consisting of multiple copies of several subunits including three enzyme activities E1, E2 and E3, required for the completion of the conversion of pyruvate to acetyl CoA (Patel and Roche 1990; FASEB J., 4: 3224-3233). E1catalyses the non-reversible removal of CO2 from pyruvate; E2 forms acetyl CoA and E3 reduces NAD to NADH. Two additional enzyme activities are associated with the complex: a specific kinase which is capable of phosphorylating E1 at three serine residues and a loosely-associated specific phosphatase which reverses the phosphorylation. Phosphorylation of a single one of the three serine residues renders the E1 inactive. The proportion of the PDH in its active (dephosphorylated) state is determined by a balance between the activity of the kinase and phosphatase. The activity of the kinase may be regulated in vivo by the relative concentrations of metabolic substrates such as NAD/NADH, CoA/acetylCoA and adenine diphosphate (ADP)/ATP as well as by the availability of pyruvate itself.
European Patent Publication No. 617010 describes compounds which are capable of relaxing bladder smooth muscle and which may be used in the treatment of urge incontinence. Whilst the compounds of the present invention fall within the general structural disclosure of EP 0617010, we have found, surprisingly, that the compounds of the present invention are very good at elevating PDH activity, a property nowhere disclosed in EP 0617010.
The present invention is based on the surprising discovery that certain compounds elevate PDH activity, a property of value in the treatment of disease states associated with disorders of glucose utilisation such as diabetes mellitus, obesity, (Curto et al., 1997; Int. J. Obes. 21: 1137-1142), and lactic acidaemia. Additionally the compounds may be expected to have utility in diseases where supply of energy-rich substrates to tissues is limiting such as peripheral vascular disease, (including intermittent claudication), cardiac failure and certain cardiac myopathies, muscle weakness, hyperlipidaemias and atherosclerosis (Stacpoole et al., 1978; N. Engl. J. Med. 298: 526-530). A compound that activates PDH may also be useful in treating Alzheimer disease (AD) (J Neural Transm (1998) 105: 855-870).
According to one aspect of the present invention there is provided the use of a compound of the formula (I): 
wherein:
ring C is phenyl or carbon-linked heteroaryl selected from pyridyl, pyrazinyl, pyrimidinyl and pyridazinyl; wherein said phenyl or heteroaryl is substituted on carbon at one or both positions meta to the position of Axe2x80x94B attachment or on carbon at the position para to the position of Axe2x80x94B attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy, trifluoromethylthio and a group ArY; and further, wherein said phenyl or heteroaryl is substituted on carbon at any remaining meta position(s) or para position by a group or groups independently selected from hydrogen, (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (1-4C)alkenyloxy, cyano, nitro, halo, hydroxy and trifluoromethylthio; in which
Ar is selected from the group consisting of phenyl, a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said phenyl or heteroaryl ring Ar is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio;
Y is selected from carbonyl, sulphinyl and sulphonyl;
Axe2x80x94B is selected from NHCO, OCH2, SCH2, NHCH2, trans-vinylene, and ethynylene;
R1 is linked to ring C at a carbon ortho to the position of Axe2x80x94B attachment and is selected from the group consisting of (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo, trifluoromethylthio and hydroxy;
n is 1 or 2;
R2 and R3 are independently (1-3C)alkyl optionally substituted by from 1 to 2k+1 atoms selected from fluoro and chloro wherein k is the number of carbon atoms in the said (1-3C)alkyl, provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a 3-5 membered cycloalkyl ring optionally substituted by from 1 to 2 mxe2x88x922 fluorine atoms wherein m is the number of carbon atoms in said ring;
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I);
and a pharmaceutically acceptable salt of said compound or said ester;
provided said compound is not
N-(4-benzoyl-2,6-dimethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide, in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Preferably ring C is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted as defined hereinbefore.
More preferably ring C is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy, trifluoromethylthio and a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
Particularly ring C is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy and a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
More particularly ring C is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group selected from cyano, trifluoromethyl, nitro and a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
Particularly preferred ring C is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
Especially ring C is phenyl and is substituted as defined hereinbefore.
More especially ring C is phenyl wherein said phenyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy and a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
Particularly preferred ring C is phenyl wherein said phenyl is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
More especially ring C is phenyl which is substituted on carbon at the position para to the position of Axe2x80x94B attachment by a group ArY (wherein Axe2x80x94B and ArY are as defined hereinbefore).
In one embodiment of the present invention Ar is phenyl, 4-pyridyl, 2-thienyl, 2-pyrimidyl, 2-imidazolyl, 2-pyridyl, 3-pyridyl, 2-thiazolyl or 3-furyl and is optionally substituted as defined hereinbefore.
In a further embodiment of the present invention Ar is phenyl (optionally substituted with fluoro, methoxy, bromo, trifluoromethyl, nitro or methyl), or Ar is 4-pyridyl, 5-methylthien-2-yl, 2-pyrimidyl, 2-imidazolyl, 2-pyridyl, 3-pyridyl, 2-thiazolyl or 2-methylfur-3-yl.
In another embodiment of the present invention Ar is phenyl or 4-pyridyl and is optionally substituted as defined hereinbefore.
In an alternative embodiment of the present invention Ar is phenyl and is optionally substituted as defined hereinbefore.
In an additional aspect of the invention Ar is selected from the group consisting of a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said heteroaryl ring (Ar) is optionally substituted as defined hereinbefore.
In one aspect of the invention, preferably Y is sulphinyl or sulphonyl.
Preferably Axe2x80x94B is NHCO or ethynylene.
More preferably Axe2x80x94B is NHCO.
Preferably R1 is selected from the group consisting of (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, cyano, nitro, halo and hydroxy.
More preferably R1 is selected from the group consisting of (1-2C)alkyl, (1-2C)alkoxy, cyano, nitro, halo and hydroxy.
Particularly R1 is selected from methyl, methoxy, nitro, fluoro, chloro, bromo and hydroxy.
More particularly R1 is selected from methoxy, nitro, fluoro, chloro, bromo and hydroxy.
Especially R1 is fluoro, chloro or bromo.
Preferably n is 1.
Preferably R2 and R3 are independently (1-3C)alkyl optionally substituted by from 1 to 2k+1 atoms selected from fluoro and chloro, wherein k is the number of carbon atoms in the said (1-3C)alkyl, provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
More preferably R2 and R3 are independently (1-2C)alkyl optionally substituted by from 1 to 2k+1 fluorine atoms, wherein k is the number of carbon atoms in the said (1-2C)alkyl, provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a Cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
Particularly R2 and R3 are independently methyl, fluoromethyl, difluoromethyl, Trifluoromethyl, 2,2,2-trifluoroethyl and perfluoroethyl provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
More particularly R2 and R3 are independently methyl, fluoromethyl, difluoromethyl and trifluoromethyl, provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
In another aspect of the invention preferably R2 and R3 are independently methyl, ethyl, difluoromethyl, trifluoromethyl provided that R2 and R3 are not both methyl; or
R2 and R3, together with the carbon atom to which they are attached, form a cyclopropane ring.
In a further aspect of the invention preferably one of R2 and R3 is methyl and the other is trifluoromethyl.
Where applicable, the R-configuration at the carbinol centre generally represents a preferred stereochemistry for compounds of formula (I).
According to another aspect of the present invention there is provided the use of a compound of the formula (Ia): 
wherein:
ring Ca is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of Aaxe2x80x94Ba attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy, trifluoromethylthio and a group AraYa (wherein Ara is selected from the group consisting of phenyl, a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said phenyl or heteroaryl ring Ar is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio, Ya is selected from carbonyl, sulphinyl and sulphonyl and Aaxe2x80x94Ba is selected from NHCO, OCH2, SCH2, NHCH2, trans-vinylene, and ethynylene);
R1a is linked to ring C at a carbon ortho to the position of Aaxe2x80x94Ba attachment and is selected from the group consisting of (1xe2x88x9d4C)alkyl, (1-4C)haloalkyl, (1-4AC)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo, trifluoromethylthio and hydroxy;
na is 1;
R2a and R3a are independently (1-3C)alkyl optionally substituted by from 1 to 2k+1 atoms selected from fluoro and chloro, wherein k is the number of carbon atoms in the said (1-3C)alkyl, provided that R2a and R3a are not both methyl; or
R2a and R3a, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms;
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ia);
and a pharmaceutically acceptable salt of said compound or said ester;
in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Where applicable, the R-configuration generally represents a preferred stereochemistry or compounds of formula (Ia).
According to another aspect of the present invention there is provided the use of a compound of the formula (Ib): 
wherein:
ring Cb is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of NHCO attachment by a group selected from cyano, trifluoromethyl, nitro, trifluoromethoxy, trifluoromethylthio and a group ArbYb (wherein Arb is selected from the group consisting of phenyl, a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said phenyl or heteroaryl ring Arb is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio and Yb is selected from carbonyl, sulphinyl and sulphonyl);
R1b is linked at a carbon ortho to the position of NHCO attachment and is selected from the group consisting of (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo, trifluoromethylthio and hydroxy;
nb is 1;
R2b and R3b are independently (1-2C)alkyl optionally substituted by from 1 to 2k+1 fluorine atoms, wherein k is the number of carbon atoms in the said (1-2C)alkyl, provided that R2b and R3b are not both methyl; or
R2b and R3b, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms;
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ib);
and a pharmaceutically acceptable salt of said compound or said ester;
in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Where applicable, the R-configuration generally represents a preferred stereochemistry for compounds of formula (Ib).
According to another aspect of the present invention there is provided the use of a compound of the formula (Ic): 
wherein:
ring Cc is phenyl or carbon-linked pyridyl wherein said phenyl or pyridyl is substituted on carbon at the position para to the position of NHCO attachment by a group ArcYc (wherein Arc is selected from the group consisting of phenyl, a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said phenyl or heteroaryl ring Arc is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio, and Yc is selected from carbonyl, sulphinyl and sulphonyl);
R1c is linked at a carbon ortho to the position of NHCO attachment and is selected from the group consisting of (1-2C)alkyl, (1-2C)alkoxy, cyano, nitro, halo and hydroxy;
R2c and R3c are independently methyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, and perfluoroethyl, provided that R2c and R3c are not both methyl; or
R2c and R3c, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms;
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ic);
and a pharmaceutically acceptable salt of said compound or said ester;
in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Where applicable, the R-configuration generally represents a preferred stereochemistry for compounds of formula (Ic).
According to another aspect of the present invention there is provided the use of a compound of the formula (Id): 
wherein:
Ard is selected from the group consisting of phenyl, a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said phenyl or heteroaryl ring Ard is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio and Yd is selected from carbonyl, sulphinyl and sulphonyl;
R1d is linked at a carbon ortho to the position of NHCO attachment and is selected from the group consisting of methoxy, nitro, fluoro, chloro, bromo and hydroxy;
R2d and R3d are independently methyl, fluoromethyl, difluoromethyl, trifluoromethyl, provided that R2d and R3d are not both methyl; or
R2d and R3d, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Id);
and a pharmaceutically acceptable salt of said compound or said ester;
in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Where applicable, the R-configuration generally represents a preferred stereochemistry for compounds of formula (Id).
Many of the compounds of the present invention are novel and as such are provided as a further feature of the present invention.
According to another aspect of the present invention there is provided a compound of the formula (I): 
wherein ring C, R1, n, Axe2x80x94B, R2 and R3 are as defined hereinbefore,
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I);
and a pharmaceutically acceptable salt of said compound or said ester;
provided said compound is not selected from
N-(4-benzoyl-2,6-dimethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-fluorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(6-chloro-3-phenylsulfonylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-methoxy-4-(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-2-methyl-N-[2-nitro-4-(phenylsulfonyl)phenyl]-3,3,3-trifluoropropanamide,
S-(xe2x88x92)-N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-chlorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-bromophenyl)-3,3-difluoro-2-(difluoromethyl)-2-hydroxypropanamide,
N-(4-benzoyl-2-bromophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-cyanophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methoxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-hydroxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-hydroxy-4(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2,6-dimethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(2-Fluoro-5-nitrophenyl)-2-hydroxy-2-trifluoromethylbutanamide,
N-(2-Fluoro-5-nitrophenyl)-2-hydroxy-2-difluoromethyl-3,3-difluoropropanamide, and
3-Hydroxy-3-trifluoromethyl-1-(2-chloro-5-trifluoromethylphenyl)-4,4,4-trifluorobut-1-yne,
and pharmaceutically acceptable in vivo cleavable esters of said compounds,
and pharmaceutically acceptable salts of said compounds and said esters.
According to another aspect of the present invention there is provided a compound of the formula (Ia): 
wherein:
ring Ca, R1a, na, Aaxe2x80x94Ba, R2a and R3a are as defined hereinbefore, and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ia); and a pharmaceutically acceptable salt of said compound or said ester; provided said compound is not selected from:
N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-fluorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-methoxy-4-(4-pyridyl-sulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-2-methyl-N-[2-nitro-4(phenyl-sulfonyl)phenyl]-3,3,3-trifluoropropanamide,
S-(xe2x88x92)-N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-chlorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-bromophenyl)-3,3-difluoro-2-(difluoromethyl)-2-hydroxypropanamide,
N-(4-benzoyl-2-bromophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-cyanophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methoxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-hydroxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide and
2-hydroxy-N-[2-hydroxy-4-(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide
and pharmaceutically acceptable in vivo cleavable esters of said compounds,
and pharmaceutically acceptable salts of said compounds and said esters.
According to another aspect of the present invention there is provided a compound of the formula (Ib): 
wherein:
Cb, R1b, R2b, R3b and nb are as defined hereinbefore,
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ib);
and a pharmaceutically acceptable salt of said compound or said ester;
provided said compound is not selected from:
N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-fluorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-methoxy-4-(4-pyridyl-sulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-2-methyl-N-[2-nitro-4-(phenyl-sulfonyl)phenyl]-3,3,3-trifluoropropanamide,
S-(xe2x88x92)-N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl -3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-chlorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4benzoyl-2-bromophenyl)-3,3-difluoro-2-(difluoromethyl)-2-hydroxypropanamide,
N-(4benzoyl-2-bromophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-cyanophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methoxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-hydroxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide and
2-hydroxy-N-[2-hydroxy-4(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
and pharmaceutically acceptable in vivo cleavable esters of said compounds,
and pharmaceutically acceptable salts of said compounds and said esters.
According to another aspect of the present invention there is provided a compound of the formula (Ic): 
wherein:
Cc, R1c, R2c, R3c, Arc and Yc are as defined hereinbefore,
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ic);
and a pharmaceutically acceptable salt of said compound or said ester;
provided said compound is not selected from:
N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyll-2-fluorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-methoxy-4-(4-pyridyl-sulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-2-methyl-N-[2-nitro-4-(phenyl-sulfonyl)phenyl]-3,3,3-trifluoropropanamide,
S-(xe2x88x92)-N-(4-benzoyl-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-chlorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-bromophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-cyanophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methoxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-hydroxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide and
2-hydroxy-N-[2-hydroxy-4-(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
and pharmaceutically acceptable in vivo cleavable esters of said compounds,
and pharmaceutically acceptable salts of said compounds and said esters.
According to another aspect of the present invention there is provided a compound of the formula (Id): 
wherein:
R1d, R2d, R3d, Ard and Yd are as defined hereinbefore,
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Id);
and a pharmaceutically acceptable salt of said compound or said ester;
provided said compound is not selected from:
N-(4-benzoyl-2-fluorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-N-[2-methoxy-4-(4-pyridyl-sulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
2-hydroxy-2-methyl-N-[2-nitro-4-(phenyl-sulfonyl)phenyl]-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-chlorophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-bromophenyl)-3,3-difluoro-2-(difluoromethyl)-2-hydroxypropanamide,
N-(4-benzoyl-2-bromophenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-benzoyl-2-methoxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-benzoyl-2-hydroxyphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide and
2-hydroxy-N-[2-hydroxy-4-(4-pyridylsulfonyl)phenyl]-2-methyl-3,3,3-trifluoropropanamide,
and pharmaceutically acceptable in vivo cleavable esters of said compounds,
and pharmaceutically acceptable salts of said compounds and said esters.
According to another aspect of the present invention there is provided a compound of the formula (Ie): 
wherein:
R1e is fluoro, chloro or bromo;
m is 1 or 2;
Are is selected from the group consisting of a carbon-linked six-membered heteroaryl ring containing 1-2 nitrogen atoms and a carbon-linked five-membered heteroaryl ring containing from 1-2 heteroatoms selected from nitrogen, oxygen, and sulphur; wherein said heteroaryl ring is optionally substituted at carbon, with 1-4 substituents selected from (1-4C)alkyl, (1-4C)haloalkyl, (1-4C)alkoxy, (1-4C)haloalkoxy, (2-4C)alkenyloxy, cyano, nitro, halo and trifluoromethylthio;
and a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ie),
and a pharmaceutically acceptable salt of said compound or said ester.
The compounds of the present invention which are of the R-configuration at the carbinol centre form a preferred group of compounds of the present invention. According to another aspect of the present invention there is provided the use of a compound selected from Examples 1, 2, 7, 8 and 10-15 and a pharmaceutically acceptable in vivo cleavable ester of said compound, and a pharmaceutically acceptable salt of said compound or said ester in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
According to a preferred aspect of the present invention there is provided the use of a compound selected from Examples 2 and 10-14 and a pharmaceutically acceptable in vivo cleavable ester of said compound, and a pharmaceutically acceptable salt of said compound or said ester, in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
According to another preferred aspect of the present invention there is provided the use of Example 1 and a pharmaceutically acceptable in vivo cleavable ester of said compound, and a pharmaceutically acceptable salt of said compound or said ester, in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
According to a more preferred aspect of the present invention there is provided the use of a compound selected from Examples 7, 8 and 15 and a pharmaceutically acceptable in vivo cleavable ester of said compound, and a pharmaceutically acceptable salt of said compound or said ester, in the manufacture of a medicament for use in the elevation of PDH activity in warm-blooded animals such as humans.
Compounds of the invention include:
(S)-2-hydroxy-2-methyl-N-(2-nitro-4-phenylsulphonylphenyl)-3,3,3-trifluoropropanamide,
(S)-N-(2-chloro-4-phenylsulphonylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-cyano-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-cyano-2-trifluoromethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide, and Examples 3, 4, 9, 16-26, 34, 35, 40-44 and 48-52 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
Preferred compounds are:
N-(4-cyano-2-methylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide,
N-(4-cyano-2-trifluoromethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide, and Examples 3, 4, 26 and 49-52 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
More preferred compounds are Examples 22, 24 and 55 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
Particularly preferred compounds are Examples 34 and 35 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
More particularly preferred compounds are Examples 9, 16, 17, 19-21, 23, 40-44 and 48 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
Another more particularly preferred compound of the invention is Example 18 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
Another more particularly preferred compound of the invention is Example 16 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
In another aspect of the invention preferred compounds of the invention include any one of Examples 3-6, 9, and 16-61 and pharmaceutically acceptable in vivo cleavable ester of said compounds and a pharmaceutically acceptable salt of said compounds or said esters.
In a further aspect of the invention preferred compounds of formula (I) or (Ie) include Examples 28, 47 and 55-61 and a pharmaceutically acceptable in vivo cleavable ester of said compound; and a pharmaceutically acceptable salt of said compound or said ester.
Preferred aspects of the invention are those which relate to the compound or a pharmaceutically acceptable salt thereof.
For the avoidance of doubt it is to be understood that where in this specification a group is qualified by xe2x80x98hereinbefore definedxe2x80x99 or xe2x80x98defined hereinbeforexe2x80x99 the said group encompasses the first occurring and broadest definition as well as each and all of the preferred definitions for that group.
In this specification the term xe2x80x9calkylxe2x80x9d includes both straight and branched chain alkyl groups but references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight chain version only. An analogous convention applies to other generic terms. Unless otherwise stated the term xe2x80x9calkylxe2x80x9d advantageously refers to chains with 1-6 carbon atoms, preferably 1-4 carbon atoms.
In this specification the term xe2x80x9calkoxyxe2x80x9d means an alkyl group as defined hereinbefore linked to an oxygen atom.
In this specification the term xe2x80x9carylxe2x80x9d includes C6-10 aromatic groups which may, if desired, carry one or more substituents selected from halogeno, alkyl, alkoxy, cyano, nitro or trifluoromethyl (wherein alkyl and alkoxy are as hereinbefore defined). The term xe2x80x9caryloxyxe2x80x9d means an aryl group as defined hereinbefore linked to an oxygen atom.
Within the present invention it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism and that the formulae drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the invention encompasses any tautomeric form which elevates PDH activity and is not to be limited merely to any one tautomeric form utilised within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been possible to show graphically herein.
It will be appreciated by those skilled in the art that certain compounds of formula (I) contain an asymmetrically substituted carbon and/or sulphur atom, and accordingly may exist in, and be isolated in, optically-active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic or stereoisomeric form, or mixtures thereof, which form possesses properties useful in the elevation of PDH activity, it being well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, by enzymatic resolution, (for example WO 9738124), by biotransformation, or by chromatographic separation using a chiral stationary phase) and how to determine efficacy for the elevation of PDH activity by the standard tests described hereinafter.
It is also to be understood that certain compounds of the formula (I) and salts thereof can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms which elevate PDH activity.
A compound of the formula (I), or salt thereof, and other compounds of the invention (as hereinafter defined) may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes include, for example, those illustrated in European Patent Applications, Publication Nos. 0524781, 0617010, 0625516, and in GB 2278054, WO 9323358 and WO 9738124.
Such processes, are provided as a further feature of the invention and are as described hereinafter, wherein, unless specified otherwise, Ar, Y, ring C, Axe2x80x94B, R1, R2, R3 and n are as defined hereinbefore. Necessary starting materials may be obtained by standard procedures of organic chemistry. The preparation of such starting materials is illustrated within the accompanying non-limiting Examples. Alternatively necessary starting materials are obtainable by analogous procedures to those illustrated which are within the ordinary skill of an organic chemist.
Thus the following processes constitute further features of the present invention. Compounds of the formula (I) and salts thereof may be prepared:
(a) by deprotecting a protected compound having formula (II): 
xe2x80x83wherein xe2x80x9cPgxe2x80x9d is a suitable alcohol protecting group such as for example a benzyl, acetate or silyl protecting group; examples of suitable reagents for deprotecting a compound of formula (II) when Pg is benzyl are (1) hydrogen in the presence of palladium-on-carbon catalyst, i.e. hydrogenolysis, or (2) hydrogen bromide or iodide; when Pg is acetate diluted inorganic base, for example aqueous lithium-, sodium- or potassium-hydroxide; and when Pg is a silyl protecting group are (1) tetrabutylammonium fluoride, or (2) aqueous hydrofluoric acid. The reaction can be conducted in a suitable solvent such as ethanol, methanol, acetonitrile, or dimethyl sulfoxide (DMSO) and may conveniently be performed at a temperature in the range of xe2x88x9240 to 100xc2x0 C.;
(b) when Y is carbonyl: by oxidizing a corresponding alcohol of formula (III): 
xe2x80x83wherein ring D1 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by ArCH(OH). Oxidizing agents such as pyridinium dichromate and solvents such as methanol and dichloromethane, respectively, may be employed;
(c) by deprotecting a corresponding compound of formula (I) wherein in place of Y is a ketal protected carbonyl, for example by deprotecting a compound of formula (IV): 
xe2x80x83wherein ring D2 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by Ar(xe2x80x94Cxe2x80x94Oxe2x80x94(CH2)3xe2x80x94Oxe2x80x94)xe2x80x94. A saturated aqueous acid such as oxalic or a mineral acid such as hydrochloric acid or sulphuric acid may conveniently be employed. The reaction may conveniently be performed at a temperature in the range of 0 to 100xc2x0 C. in a solvent such as a lower alcohol (e.g., methanol or ethanol), or mixtures of solvent pairs such as water/dichloromethane, water/tetrahydrofuran, and water/acetone;
(d) by treating a compound of formula (V): 
xe2x80x83wherein ring D3 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by G1 and G1 is defined as a leaving group such as for example bromo, iodo or triflate, with a tin compound having the formula (R6)p1Sn(Ar)p2 (wherein R6 is (1-4C)alkyl such as methyl or butyl and p1+p2=4) and carbon monoxide to effect carbonylative coupling, in the presence of a suitable catalyst such as bis(triphenylphosphine)palladium dichloride. The reaction may conveniently be performed at a temperature in the range of 0 to 100xc2x0 C. and in a solvent such as tetrahydrofuran, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, or DMSO;
(e) by treating a compound of formula (V) wherein G1 is a leaving group such as for example bromo, iodo or triflate, with an aluminium compound having the formula (R6)p3Al(Ar)p4 (wherein R6 is (1-4C)alkyl such as methyl or butyl and p3+p4=3) and carbon monoxide to effect carbonylative coupling, in the presence of a suitable catalyst such as bis(triphenylphosphine)palladium dichloride. The reaction may conveniently be performed at a temperature in the range of 0 to 100xc2x0 C. and in a solvent such as diethyl ether, benzene, toluene, or tetrahydrofuran;
(f) when Y is sulfonyl: by treating a compound of formula (V), wherein G1 is a leaving group such as for example bromo or iodo, especially iodo, with a compound of formula ArSO2xe2x88x92Na+ in the presence of a Cu(I) catalyst such as cuprous iodide. The reaction may conveniently be performed at a temperature in the range of 30 to 200xc2x0 C. and in a solvent such as N,N-dimethylformamide, 1,3-dimethyl-3,4,5,6-tetra-hydro-2(1H)-pyrimidinone, DMSO, or ethylene glycol;
(g) when Y is sulfinyl or sulfonyl and Axe2x80x94B is any of the values defined above, except SCH2 or NHCH2: by oxidizing a compound of formula (V) wherein G1 is ArS. Suitable oxidizing agents include potassium permanganate, OXONE (trade mark of E. I. du Pont de Nemours and Co.,Inc), sodium periodate, tert-butyl hydroperoxide (as solution in toluene), 3-chloroperoxybenzoic acid and hydrogen peroxide. The reaction may be conducted in a suitable solvent such as diethyl ether, dichloromethane, methanol, ethanol, water, acetic acid, or mixtures of two or more of the aforementioned. The reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 70xc2x0 C.;
(h) when Axe2x80x94B is NHCO: by coupling a compound of formula (VI): 
xe2x80x83wherein J is NH2 with an acid of formula (VII): 
xe2x80x83wherein G2 is a hydroxy group. The reaction can be conducted in a suitable solvent and in the presence of a suitable coupling reagent. Suitable coupling reagents, for use in situ and generally known in the art as standard peptide coupling reagents, can be employed, for example thionyl chloride, oxalyl chloride, carbonyldiimidazole and dicyclohexyl-carbodiimide, optionally in the presence of a catalyst such as 4-dimethylaminopyridine or 4-pyrrolidinopyridine. A suitable base for this process is for example triethylamine, pyridine, or 2,6-di-alkyl-pyridines such as for example 2,6-lutidine or 2,6-di-tert-butylpyridine. Suitable solvents include dimethylacetamide, dichloromethane, benzene, tetrahydrofuran, and dimethylformamide. The coupling reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 40xc2x0 C. The reaction may also be effected using an acid of formula (VII) wherein G2 is protected hydroxy to give a compound of the formula (II) followed by deprotection, (which may be effected by a process as described in (a) above), to give a compound of formula (I);
(i) by coupling a compound of formula (VI) wherein J is NH2 with an activated acid derivative of an acid of formula (VII), wherein G2 is a hydroxy group, such as for example stable acid chlorides, acid anhydrides, or phenylesters in the presence of a base such as for example triethylamine, pyridine, or 2,6-di-alkyl-pyridines such as for example 2,6-lutidine or 2,6-di-tert-butylpyridine. Suitable solvents include dimethylacetamide, dichloromethane, benzene, tetrahydrofuran, and dimethylformamide. The coupling reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 40xc2x0 C. The reaction may also be effected using an activated acid derivative of formula (VII) wherein G2 is protected hydroxy to give a compound of the formula (II) followed by deprotection, (which may be effected by a process as described in (a) above), to give a compound of formula (I);
(j) by reacting an amide of formula (VIII): 
xe2x80x83with a base sufficiently basic (e.g., a lithium dialkylamide such as lithium diisopropyl amide) to yield an amide dianion, followed by treatment of the dianion with oxygen in the presence of a reducing agent (e.g., such as triphenylphosphine). The sequence of reactions may conveniently be performed at a temperature in the range of xe2x88x92100 to xe2x88x9220xc2x0 C., preferably at a temperature in the range of xe2x88x9220 to 50xc2x0 C., in a suitable solvent such as tetrahydrofuran or diethyl ether;
(k) by reacting a corresponding compound of formula (IX): 
xe2x80x83wherein Hal indicates a halogen substituent (e.g., the corresponding chloride), with a corresponding alkali metal amide dianion having formula (X); 
xe2x80x83wherein M is an alkali metal such as sodium or lithium. The reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 100xc2x0 C. and in a suitable solvent such as dimethylformamide, DMSO, or tetrahydrofuran;
(l) when Y is sulfonyl and R2xe2x95x90R3: by reacting a corresponding compound of formula (XI): 
xe2x80x83wherein OR4 is an alcohol residue such as for example methoxy or ethoxy, with a Grignard compound of formula R2MgBr or R2MgCl. The reaction may conveniently be performed at a temperature in the range of xe2x88x92100 to 20xc2x0 C., preferably at a temperature in the range of xe2x88x9220 to 20xc2x0 C., in a suitable solvent such as tetrahydrofuran or diethyl ether;
(m) when Y is sulfonyl: by treating a corresponding compound of formula (XII): 
xe2x80x83with a compound of formula R2M wherein M is an alkali metal (such as lithium) or a Grignard compound of formula R2MgBr or R2MgCl. The reaction may conveniently be performed at a temperature in the range of xe2x88x92100 to 0xc2x0 C. and in a solvent such as tetrahydrofuran, diethyl ether, or 1,2-dimethoxyethane;
xe2x80x83(n) when Axe2x80x94B is ethynylene: by coupling a corresponding compound of formula (XIII): 
xe2x80x83wherein L is a leaving group such as bromo, iodo or triflate, with a corresponding acetylene of formula (XIV): 
xe2x80x83in the presence of a catalyst such as a combination of cuprous iodide and bis(triphenyl-phosphine)palladium dichloride or palladium(II) acetate to give a compound of formula (II). The reaction can be conducted in an inert solvent such as tetrahydrofuran, benzene, or toluene, or in a basic solvent such as diethylamine (DEA) or triethylamine (TEA), and at a temperature in the range of xe2x88x9220 to 110xc2x0 C. The compound of formula (II) so formed can be deprotected as described in (a) hereinbefore;
(o) by reacting a corresponding alkyne of formula (XV): 
xe2x80x83with a base such as lithium diisopropylamide (LDA), n-butyllithium or tert-butyllithium, followed by treatment with a ketone of formula R3xe2x80x94COxe2x80x94R2. The reaction may conveniently be performed at a temperature in the range of xe2x88x92100 to 40xc2x0 C. preferably at a temperature in the range of xe2x88x9270 to xe2x88x9240xc2x0 C. and in a solvent such as tetrahydrofuran, diethyl ether, or 1,2-dimethoxyethane;
(p) when Axe2x80x94B is trans-vinylene: by reducing a corresponding acetylene of formula (XVI): 
xe2x80x83with a suitable reducing agent, for example lithium aluminum hydride. The reaction can be conducted in a suitable solvent such as tetrahydrofuran or diethyl ether, and at a temperature in the range of 0 to 50xc2x0 C.;
(q) by dehydration of a diol of formula (XVII): 
xe2x80x83in the presence of an acid catalyst (for example p-toluenesulfonic acid), neat or with a solvent such as toluene or dichloromethane, and at a temperature in the range of 0 to 200xc2x0 C. preferably a temperature in the range of 20 to 100xc2x0 C.;
(r) by base catalysed opening of an epoxide of formula (XVIII): 
xe2x80x83The opening may be carried out in a suitable organic solvent such as for example, ethers, alcohols, or toluene, of which ethers and toluene are preferred, especially ethers such as tetrahydrofuran. Suitable bases include for example potassium tert-butoxide or sodium hydride. A basic aqueous solution may conveniently be employed. A preferred base is aqueous sodium hydroxide. The opening may be carried out at a temperature in the range of xe2x88x9250xc2x0 C. to 100xc2x0 C., preferably at a temperature in the range of 0 to 50xc2x0 C., such as for example room temperature;
(s) when Axe2x80x94B is NHCH2: by reducing a corresponding compound of formula (I) in which Axe2x80x94B is NHCO, with a suitable reducing agent such as lithium aluminum hydride or borane. The reaction can conveniently be carried out at a temperature in the range of 0xc2x0 C. to reflux, in solvents such as for example diethyl ether, tetrahydrofuran, or 1,2-dimethoxyethane;
(t) when Axe2x80x94B is OCH2, SCH2 or NHCH2: by reacting an ethylene oxide of formula (XIX): 
with a corresponding compound of formula (VI) (wherein J is, correspondingly, oxygen, sulphur or NH), in the presence of a base such as for example sodium hydride. The reaction can be conducted at reflux in a solvent such as dichloromethane;
(u) by heating a corresponding compound of formula (XX): 
wherein R5 is correspondingly, OH, NH2 or SH in the presence of a base such as for example an alkali metal hydride at a temperature in the range of 20xc2x0 C. to about reflux, in a solvent such as N,N-dimethylformamide;
(v) when Y is sulfonyl, Axe2x80x94B is trans-vinylene or ethynylene, and R2xe2x95x90R3: for a compound of formula (I) which bears a hydroxy substituent on an aryl or heteroaryl group, by cleaving the alkyl ether or acyloxy ester of a corresponding compound of formula (I) which bears a lower alkoxy or lower acyloxy substituent on an aryl or heteroaryl group. Convenient methods include, for example, the cleavage of a methoxy group using boron tribromide and the cleavage of a tert-butoxy group using trifluoroacetic acid;
(w) a compound of formula (I), wherein ring C is substituted by ArSO2, Axe2x80x94B is NHCO and R1 is ortho-nitro may be made from a compound of formula (XXI): 
wherein ring D4 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by ArSO2, (1) on treatment with nitric acid, thereafter (2) treating the nitrated compound under mild alkaline conditions (i.e., employing a base such as lithium hydroxide) to aid the cleavage of the acetate group to yield the desired amine, which can be converted to a compound of formula (II), wherein Axe2x80x94B is NHCO in a manner analogous to that described in procedure (h) or (i) hereinbefore for making an amide, that is, by coupling the amine formed with a corresponding acid or activated acid derivative of formula (VII). The resulting compound of formula (II) may then be deprotected by a process as described in (a) hereinbefore to give a compound of formula (I); and
(x) a compound of formula (I), wherein ring C is substituted by ArCxe2x95x90O and Axe2x80x94B is NHCO, may be made by (1) acylation of a compound of formula (VI) wherein J is NH2. Acylating reagents such as benzoic acids, or derivatives thereof, may be employed in the presence of the appropriate activating reagent such as for example polyphosphoric acid. The reaction may conveniently be performed at a temperature in the range of 0 to 200xc2x0 C. employing a solvent such as N,N-dimethylformamide, 1,3-dimethyl-3,4,5,6-tetra-hydro-2(1H)-pyrimidinone, DMSO, or ethylene glycol if required, followed by (2) the formation of an amide as described in (i) or (j) hereinbefore (Staskum, B., J. Org. Chem. (1964), 29, 2856-2860; Ohnmacht C., J. Med. Chem. (1996), 39, 4592-4601). The compound of formula (II) so formed may then be deprotected for example as described in (a) hereinbefore.
If not commercially available, the necessary starting materials for the procedures such as those described above may be made by procedures which are selected from standard organic chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the above described procedure or the procedures described in the examples. In the discussion which follows, xe2x80x9cArxe2x80x9d refers to an unsubstituted or substituted phenyl group or heteroaryl group as previously defined.
In general, a compound of formula (II), wherein AB is OCH2, SCH2 or NHCH2 may be made by treating a corresponding compound of formula (VI) (wherein J is, correspondingly, oxygen, sulphur, or NH) with a corresponding compound of formula (XXII): 
wherein Pg is a protective group such as silyl and X is a suitable leaving group such as for example mesylate or triflate, in the presence of a base such as an alkali metal hydride (e.g., sodium hydride), in a solvent such as tetrahydrofuran, N,N-dimethylformamide, DMSO, or 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, and at a temperature of about 20xc2x0 C. to about reflux.
Compounds of formulae (II), (III), (VIII) and (X), wherein Axe2x80x94B is NHCO, may be made in a manner analogous to that described in (h) or (i) above for making an amide of formula (II); that is, by coupling a corresponding amine with a corresponding acid. Thus, to make a protected amide of formula (II), a corresponding amine of formula (VI) may be coupled with an acid of formula (VII) wherein the group corresponding to G2 is O. The protected acid may be made by a conventional procedure, for example by (1) esterifying an acid of formula (VII) wherein G2 is hydroxy by means of a conventional esterification procedure such as reaction with a lower alcohol (e.g., methanol) in the presence of an acid catalyst (for example sulfuric acid) if carboxylate protection is required; (2) reaction of the ester thus formed with an agent which provides the protecting group Pg, such as benzyl chloride (to provide a benzyl protecting group) or any of the conventional silylating agents known and used for such purpose (such as 2-trimethylsilylethoxymethyl chloride, SEM, in the presence of a suitable base such as sodium hydroxide or triethylamine optionally in the presence of a catalyst such as 4-dimethylaminopyridine (DMAP)); and (3) cleavage of the ester group under mild alkaline conditions (i.e., employing a base such as potassium carbonate) to yield the desired protected acid.
If the resolved acid is required it may be prepared by any of the known methods for preparation of optically-active forms (for example, by recrystallization of the chiral salt, by enzymatic resolution, (for example WO 9738124), by biotransformation, or by chromatographic separation using a chiral stationary phase). For example if an (R)-(+) resolved acid is required it may be prepared by the method of Scheme 2 in World Patent Application Publication No. WO 9738124 for preparation of the (S)-(xe2x88x92) acid, ie. using the classical resolution method described in European Patent Application Publication No. EP 0524781, also for preparation of the (S)-(xe2x88x92) acid, except that (1S,2R)-norephedrine may be used in place of(S)-(xe2x88x92)-1-phenylethylamine.
A compound of formula (II) wherein Axe2x80x94B is OCH2, SCH2 or NHCH2 may be made by protecting a corresponding alcohol made in essentially any earlier stage of synthesis, for example an alcohol of formula (V) or (VI). The alcohol may be treated with a compound which reacts to form a trialkylsilyl (e.g. trimethylsilyl) group, such as trimethylsilyl chloride or trimethylsilyl triflate, in the presence of a base such as sodium hydride, diisopropylethylamine or triethylamine. A preferred base is diisopropylethylamine. Alternatively, if an ester protecting group is desired the alcohol may be reacted with an acid chloride of formula R7COCl or an acid anhydride of formula (R7CO)2O wherein R7 is a lower alkyl or aryl group. the reaction may be conducted in a solvent such as dichloromethane, in the presence of a base such as TEA and DMAP, and at a temperature of xe2x88x9240 to about 25xc2x0 C.
A compound of formula (IV), wherein Axe2x80x94B is ethynylene, may be made by reacting a corresponding compound of formula (XXIII): 
wherein ring D5 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by Ar(xe2x80x94Cxe2x80x94Oxe2x80x94(CH2)3xe2x80x94Oxe2x80x94)xe2x80x94 with a base such as an alkyllithium (for example, butyllithium) followed by addition of a ketone having the formula R2xe2x80x94COxe2x80x94R3. The reaction may be conducted at a temperature of from about xe2x88x92100 to about 40xc2x0 C. and in a solvent such as tetrahydrofuran, dimethyl ether, or 1,2-dimethoxyethane.
A compound of formula (IV), wherein Axe2x80x94B is trans-vinylene, may be made by reducing a corresponding compound of formula (I), wherein Axe2x80x94B is ethynylene, with a suitable reducing agent such as lithium aluminium hydride or sodium bis(2-methoxyethoxy)aluminium, in a solvent such as tetrahydrofuran and at room temperature.
A compound of formula (II), wherein Axe2x80x94B is trans-vinylene, may be made by reacting a corresponding compound of formula (XXIV): 
wherein Pg is a protecting group such as for example benzyl or silyl and M is a metal such as trialkyltin (for example tributyl or trimethyl tin) or bisalkyloxyborane (for example catecholborane), with a compound of formula (IX), wherein Hal may be for example iodide or bromide, in the presence of a catalyst such as bis(triphenylphosphine)palladium dichloride or tetrakistriphenylphosphinepalladium. The reaction may conveniently be conducted in a suitable inert solvent such as a tetrahydrofuran or dimethylformamide at a temperature of from 0-150xc2x0 C.
A compound of formula (XXIV) may be made by reacting a corresponding alcohol of formula (XXV): 
with (1) an agent which provides the protecting group Pg, such as benzyl bromide (to provide a benzyl protecting group) or any of the conventional silylating agents known and used for such purpose (such as for example tert-butyl dimethylsilylchloride or triflate, in the presence of a suitable base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine optionally in the presence of a catalyst such as DMAP); (2) reaction of the protected propargylic alcohol thus formed with an agent such as catecholborane, to form the vinylborane, or trialkyltin hydride in the presence of a catalytic amount of a radical chain initiator such as for example azo-bis-isobutyronitrile (AIBN), or a strong base such as alkyllithium and copper cyanide, or a transition metal catalyst such as for example tetrakistriphenylphosphinepalladium. The reaction may conveniently be conducted in a suitable inert solvent such as a tetrahydrofuran, toluene or xylene at a temperature of from 0-150xc2x0 C.
A compound of formula (XXIII) may be made by treating the corresponding ketone with 1,3-propanediol in the presence of an acid catalyst such as p-toluenesulfonic acid (TsOH) and in a refluxing solvent such as toluene.
A compound of formula (XV) may be made by (1) treating a corresponding compound of formula (XIII) wherein L is bromo with a protected acetylene such as trimethylsilylacetylene in the presence of a catalyst such as a combination of cuprous iodide and bis(triphenylphosphine)palladium dichloride in a solvent such as diethylamine, thereby making a corresponding compound of formula (XXVI): 
wherein Axe2x80x94B is ethynylene and Ps is a silyl protecting group, followed by (2) removal of the silyl protecting group with a base such as an alkali metal (e.g. sodium hydroxide) in a solvent such as methanol.
A compound of formula (V) wherein G1 is halo, may be made by (1) treating a corresponding compound of formula (V), wherein G1 is nitro, with a reducing agent such as tin(II)chloride, in the presence of an aqueous acid such as acetic acid to obtain the corresponding amine, followed by (2) treating the amine with a combination of nitric acid and sulfuric acid to effect diazotization, and thereafter (3) treating the diazotized compound with a corresponding copper(I)halide such as for example cuprous bromide.
A compound of formula (V), wherein G1 is SH and Axe2x80x94B is NHCO can be made by (1) coupling of a compound of formula (V) wherein G1 is a leaving group such as halo or triflate with triisopropylsilanethiolate under palladium catalysis as described by Arnould et. al. in Tet. Let. (1996), 37 (26), p. 4523, followed by deprotection with tetrabutylammonium fluoride in a solvent such as tetrahydrofuran at a temperature of xe2x88x9278 to about 25xc2x0 C.; or (2) by Pummerer rearrangement as described in Tet. Let. (1984), 25 (17), p. 1753 of a compound of formula (V) wherein G1 is CH3SOxe2x80x94 and Axe2x80x94B is NHCO, which can be made from a compound of formula (V) wherein G1 is a leaving group such as halo of triflate and Axe2x80x94B is NHCO, using a palladium catalysed coupling with methanethiol as described for example in Zheng et. al. in J. Org. Chem. (1998), 63, p. 9606 followed by an oxidation of the resulting sulphide to the corresponding sulphoxide using for example tert.-butyl hydroperoxide as oxidant; or (3) reduction of a compound of formula (V), wherein G1 is SO2Cl and Axe2x80x94B is NHCO, by reducing the sulphonyl chloride using a small excess of for example triphenylphosphine in a solvent such as for example dichloromethane in the presence of a catalyst such as for example dimethylforamide, followed by an acidic workup.
A compound of formula (V), wherein G1 is SO2Cl and Axe2x80x94B is NHCO can be made by treatment with chlorosulphonic acid of a compound of formula (V), wherein G1 is H and Axe2x80x94B is NHCO, under standard conditions.
A compound of formula (XII), wherein Axe2x80x94B is ethynylene, may be made by treating a corresponding compound of formula (XXVI) wherein Ps is trimethylsilyl, with a fluoride base (for example, tetrabutylammonium fluoride (TBAF)) and an acid chloride of formula R3xe2x80x94COxe2x80x94Cl, thereby making the desired compound.
A compound of formula (XIII), wherein L is halo, may be made by treating a corresponding compound of formula (XIII), wherein L is nitro, with (1) iron dust and concentrated hydrochloric acid in 95% ethanol to reduce the nitro group and thereby form the corresponding amine; (2) the amine may then be treated for example with a nitrite (such as tert-butyl nitrite or sodium nitrite in the presence of a mineral acid) to form the corresponding diazonium salt which may in turn be treated with a copper(I) salt (such as copper(I)bromide or copper(I)chloride). The diazotization and displacement reactions may be conducted, in a solvent such as acetonitrile and at a temperature of from 0 to 25xc2x0 C.
A compound of formula (XIV) may be made by reacting a corresponding ketone having the formula R2xe2x80x94COxe2x80x94R3 with an alkali metal acetylide (for example lithium acetylide) or alkaline earth metal acetylide (for example magnesium acetylide). The reaction may be conducted in a solvent such as tetrahydrofuran, diethyl ether, or 1,2-dimethoxyethane and at a temperature of about xe2x88x92100 to about 25xc2x0 C.
A compound of formula (XV) may be made by reacting a corresponding compound of formula (XIII), wherein L is halo, with trimethylsilylacetylene in the presence of a catalyst such as a combination of bis(triphenylphosphine)palladium dichloride and copper(I)iodide in diethylamine or triethylamine, followed by treatment with a base (for example, an alkali metal hydroxide such as sodium or lithium hydroxide) in a lower alcohol as solvent to effect removal of the trimethylsilyl group.
A compound of formula (XIX) may be made by treating a corresponding ketone having the formula R2xe2x80x94COxe2x80x94R3 with the ylide derived from the reaction of a trimethylsulfonium salt (such as trimethylsulfonium iodide) with a base (such as an alkali metal hydroxide). The reaction may be conducted in a one-pot process employing a solvent such as dichloromethane.
A compound of formula (VI), wherein J is oxy, may be made by diazotizing a corresponding amine of formula (XIII), wherein L is amino, as previously discussed, and heating in dilute sulfuric acid to form the corresponding phenol. The corresponding thiophenol may be formed by reacting an excess of methanethiol in the presence of sodium hydride with a corresponding compound of formula (XIII) wherein L is a leaving group such as for example chloro.
A compound of formula (XX) wherein R5 is hydroxy, thiohydroxy or amino may be made by treating a corresponding compound of formula (XIII) wherein L is a halo group with a corresponding compound of formula (XXII), wherein X is hydroxy, thiohydroxy, or amino, and Pg is hydrogen. The reaction may conveniently be carried out in the presence of a catalyst such as copper bronze and a base such as an alkali metal hydride. The reaction may be conducted at reflux in a solvent such as tetrahydrofuran.
A compound of formula (XXII), wherein X is for example triflate, may be made by (1) esterifying an acid of formula (VII) wherein G2 is hydroxy; (2) protecting the alcohol G2, by treating with for example trimethylsilyl chloride in a solvent such as dichloromethane and at a temperature of from about xe2x88x9278 to about 25 xc2x0C.; (3) treating the protected compound thus obtained with a suitable reducing agent such as lithium aluminum hydride in a solvent such as diethyl ether or tetrahydrofuran and at a temperature of about 0 to about 25xc2x0 C., thereby reducing the carbonyl group to methylene, followed by (4) treating the reduced product with trifluoromethylsulfonic anhydride in the presence of a base such as triethylamine, in a solvent such as dichloromethane, and at a temperature in the range of about xe2x88x9278 xc2x0C. to about 25xc2x0 C.
An epoxide of formula (XVIII) may be prepared from a diol of formula (XVII) using a suitable dehydrating agent, for example bis[xcex1,xcex1-bis(trifluoromethyl)benzenemethanolato]diphenylsulphur. A diol of formula (XVII) may be prepared from a corresponding compound of formula (I), wherein Axe2x80x94B is CH2CO, by reduction. The reduction may be carried out using a suitable reducing agent, for example a hydride, such as sodium borohydride.
A corresponding compound of formula (I), wherein Axe2x80x94B is CH2CO, may be prepared from a compound of formula (XIII), wherein L is methyl, by deprotonation and treatment with an amide of formula (XXVII): 
in which R8 and R9 are each independently lower alkyl, or in which R8 and R9 when taken together with the atoms to which they are attached form a 5-7 membered ring. The deprotonation of the toluene may be carried out with a suitable base, for example lithium diisopropyl amide. The reaction may be carried out in a suitable organic solvent, for example, an ether such as tetrahydrofuran. The reaction may be carried out at a suitable temperature, for example a temperature in the range of xe2x88x9278xc2x0 C. to 100xc2x0 C.
An amide of formula (XXVII) may be prepared from an acid of formula (VII), wherein G2 is hydroxy, or an activated derivative thereof, by reaction with the corresponding amine.
A diol of formula (XVII) may be prepared by (1) treating a ketone of formula (XIII), wherein L is acetyl, with a base such as lithium diisopropylamide, lithium hexamethyldisilazide (LHMDS), or tetramethylpiperadide, in a solvent such as tetrahydrofuran, diethyl ether, or 1,2-dimethoxyethane, followed by addition of a ketone having the formula R2xe2x80x94COxe2x80x94R3 (aldol addition), and at a temperature of about xe2x88x92100 to about 25xc2x0 C., followed by (2) reduction of the carbonyl group to alcohol with a reducing agent such as sodium borohydride or lithium aluminum hydride at a temperature of from about 0 to about 25xc2x0 C.
A corresponding compound of formula (I), wherein ring C is substituted by ArS, Axe2x80x94B is NHCO and R1 is ortho-halo or ortho-hydroxy, may be made by treatment of a compound of formula (XXVIII): 
wherein ring D6 has any of the values defined hereinbefore for ring C but in which the place of one of the possible substituents on ring C is taken by ArS and wherein the amino group is in a position ortho to the nitro group, with (1) a combination of nitric acid and sulphuric acid to effect diazotization, and thereafter (2) treating the diazotized compound with a corresponding copper (I) halide such as for example cuprous bromide or chloride, or heating in dilute sulphuric acid to form the corresponding phenol, followed by (3) reduction of the nitro group with a reducing agent such as tin(II)chloride or iron dust in conjunction with concentrated acid followed by (4) amide formation as described in (h) or (i) hereinbefore. The diazotization and displacement reactions may be conducted in a solvent such as acetonitrile and at a temperature of from 0-25xc2x0 C.
A compound of formula (XXVIII) may be made for example according to procedures similar to those described in J. Med. Chem., 1975, 18, 1164.
A corresponding compound of formula (I), wherein ring C is substituted by ArS and Axe2x80x94B is NHCO may be made by treatment of a compound of formula (V), wherein G1 is a displaceable group such as halo or triflate, with a thiophenol in the presence of a catalyst such as tetrakis(triphenylphosphine)palladium(0) or cuprous chloride. The reaction may conveniently be conducted in a suitable inert solvent such as a lower alcohol or dimethylformamide and in the presence of a base such as for example sodium methoxide if required.
A corresponding compound of formula (I), wherein ring C is substituted by ArS and Axe2x80x94B is NHCO may be made by treating a compound of formula (V), wherein G1 is SH with an aromatic compound containing a displaceable group such as for example halo or triflate, in the presence of a catalyst such as tetrakis(triphenylphosphine)palladium(0) or cuprous chloride. The reaction may conveniently be conducted in a suitable inert solvent such as a lower alcohol Axe2x80x94E or dimethylformamide and in the presence of a base such as for example sodium methoxide if required at a temperature of 25-180xc2x0 C.
A corresponding compound of formula (I), wherein ring C is substituted by ArS and Axe2x80x94B is not NHCO may be made by treating a compound of formula (V), wherein G1 is an appropriate leaving group such as for example fluoro, with a compound of formula ArSH in the presence of a suitable base such as for example potassium carbonate. The reaction may conveniently be performed at a temperature in the range of 30 to 200xc2x0 C. and in a solvent such as N,N-dimethylformamide, 1,3-dimethyl-3,4,5,6-tetra-hydro-2(1H)-pyrimidinone, DMSO, or ethylene glycol.
According to a further feature of the invention, there is provided a process for preparing a compound of formula (Ie) using any one of processes a), f), g), h), i), j), k) or m); and thereafter if necessary:
i) converting a compound of the formula (I) into another compound of the formula (I);
ii) removing any protecting groups; or
iii) forming a pharmaceutically acceptable salt or in vivo cleavable ester.
It is noted that many of the starting materials for synthetic methods for intermediates as described above are commercially available and/or widely reported in the scientific literature. If not commercially available, the necessary starting materials for the procedures such as that described above may be made by procedures which are selected from standard organic chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the above described procedure or the procedures described in the examples.
In cases where compounds of formula (I) are sufficiently basic or acidic to form stable acid or basic salts, administration of the compound as a salt may be appropriate, and pharmaceutically acceptable salts may be made by conventional methods such as those described following. Examples of suitable pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, tartrate, citrate, succinate, benzoate, ascorbate, xcex1-ketoglutarate, and xcex1-glycerophosphate. Suitable inorganic salts may also be formed such as sulfate, nitrate, and hydrochloride.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound of formula (I) (or its ester) with a suitable acid affording a physiologically acceptable anion. It is also possible with most compounds of the invention to make a corresponding alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salt by treating a compound of formula (I) (and in some cases the ester) with one equivalent of an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g. the ethoxide or methoxide in aqueous medium followed by conventional purification techniques.
In vivo cleavable esters of compounds of the invention may be made by coupling with a pharmaceutically acceptable carboxylic acid or an activated derivative thereof. For example, the coupling may be carried out by treating a compound of formula (a) with an appropriate acid chloride (for example, acetyl chloride, propionyl chloride, or benzoyl chloride) or acid anhydride (for example, acetic anhydride, propionic anhydride, or benzoic anhydride) in the presence of a suitable base such as triethylamine. Those skilled in the art will appreciate that other suitable carboxylic acids (including their activated derivatives) for the formation of in vivo cleavable esters are known to the art and these are also intended to be included within the scope of the invention. Catalysts such as 4-dimethylaminopyridine may also be usefully employed.
Many of the intermediates defined herein are novel and these are provided as a further feature of the invention.
The identification of compounds which elevate PDH activity is the subject of the present invention. These properties may be assessed, for example, using one or more of the procedures set out below:
(a) In vitro Elevation of PDH activity
This assay determines the ability of a test compound to elevate PDH activity. cDNA encoding PDH kinase may be obtained by Polymerase Chain Reaction (PCR) and subsequent cloning. This may be expressed in a suitable expression system to obtain polypeptide with PDH kinase activity. For example rat PDHkinaseII (rPDHKII) obtained by expression of recombinant protein in Escherichia coli (E. Coli), was found to display PDH kinase activity.
In the case of the rPDHKII (Genbank accession number U10357) a 1.3 kb fragment encoding the protein was isolated by PCR from rat liver cDNA and cloned into a vector (for example pQE32xe2x80x94Quiagen Ltd.). The recombinant construct was transformed into E. coli (for example M15pRep4xe2x80x94Quiagen Ltd.). Recombinant clones were identified, plasmid DNA was isolated and subjected to DNA sequence analysis. One clone which had the expected nucleic acid sequence was selected for the expression work. Details of the methods for the assembly of recombinant DNA molecules and the expression of recombinant proteins in bacterial systems can be found in standard texts for example Sambrook et al, 1989, Molecular Cloningxe2x80x94A Laboratory Manual , 2nd edition, Cold Spring Harbour Laboratory Press. Other known PDH kinases for use in assays, may be cloned and expressed in a similar manner.
For expression of rPDHKII activity, E. coli strain M15pRep4 cells were transformed with the pQE32 vector containing rPDHKII cDNA. This vector incorporates a 6-His tag onto the protein at its N-terminus. E. coli were grown to an optical density of 0.6 (600 nM) and protein expression was induced by the addition of 10 xcexcM isopropylthio-xcex2-galactosidase. Cells were grown for 18 hours at 18xc2x0 C. and harvested by centrifugation. The resuspended cell paste was lysed by homogenisation and insoluble material removed by centrifugation at 24000xc3x97g for 1 hour. The 6-His tagged protein was removed from the supernatant using a nickel chelating nitrilotriacetic acid resin (Ni-NTA: Quiagen Ltd.) matrix (Quiagen) which was washed with 20 mM tris(hydroxymethyl)aminomethane-hydrogen chloride, 20 mM imidazole, 0.5 M sodium chloride pH 8.0, prior to elution of bound protein using a buffer containing 20 mM tris(hydroxymethyl)aminomethane-hydrogen chloride, 200 mM imidazole, 0.15 M sodium chloride pH 8.0. Eluted fractions containing 6-His protein were pooled and stored in aliquots at xe2x88x9280xc2x0 C. in 10% glycerol.
Each new batch of stock enzyme was titrated in the assay to determine a concentration giving approximately 90% inhibition of PDH in the conditions of the assay. For a typical batch, stock enzyme was diluted to 7.5 xcexcg/ml.
For assay of the activity of novel compounds, compounds were diluted with 10% dimethylsulphoxide (DMSO) and 10 xcexcl transferred to individual wells of 96-well assay plates. Control wells contained 20 xcexcl 10% DMSO instead of compound. 40 xcexcl Buffer containing 50 mM potassium phosphate buffer pH 7.0, 10 mM ethylene glycol-bis(xcex2-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA), 1 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride (PMSF), 0.3 mM tosyl-L-lysine chloromethyl ketone (TLCK), 2 mM dithiothreitol (DTT), recombinant rPDHKII and compounds were incubated in the presence of PDH kinase at room temperature for 45 minutes. In order to determine the maximum rate of the PDH reaction a second series of control wells were included containing 10% DMSO instead of compound and omitting rPDHKII. PDH kinase activity was then initiated by the addition of 5 xcexcM ATP, 2 mM magnesium chloride and 0.04 U/ml PDH (porcine heart PDH Sigma P7032) in a total volume of 50 xcexcl and plates incubated at ambient temperature for a further 45 minutes. The residual activity of the PDH was then determined by the addition of substrates (2.5 mM coenzyme A, 2.5 mM thiamine pyrophosphate (cocarboxylase), 2.5 mM sodium pyruvate, 6 mM NAD in a total volume of 80 xcexcl and the plates incubated for 90 minutes at ambient temperature. The production of reduced NAD (NADH) was established by measured optical density at 340 nm using a plate reading spectrophotometer. The ED50 for a test compound was determined in the usual way using results from 12 concentrations of the compound.
(b) In vitro Elevation of PDH Activity in Isolated Primary Cells
This assay determines the ability of compounds to stimulate pyruvate oxidation in primary rat hepatocytes.
Hepatocytes were isolated by the two-step collagenase digestion procedure described by Seglen (Methods Cell Biol. (1976) 13, 29-33) and plated out in 6-well culture plates (Falcon Primaria) at 600000 viable cells per well in Dulbecco""s Modified Eagles Medium (DMEM, Gibco BRL) containing 10% foetal calf serum (FCS), 10% penicillin/streptomycin (Gibco BRL) and 10% non-essential amino acids (NEAA, Gibco BRL). After 4 hours incubation at 37xc2x0 C. in 5% CO2, the medium was replaced with Minimum Essential Medium (MEM, Gibco BRL) containing NEAA and penicillin/streptomycin as above in addition to 10 nM dexamethasone and 10 nM insulin.
The following day cells were washed with phosphate buffered saline (PBS) and medium replaced with 1 ml HEPES-buffered Krebs solution (25 mM HEPES, 0.15M sodium chloride, 25 mM sodium hydrogen carbonate, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium sulphate, 1 mM potassium dihydrogen phosphate) containing the compound to be tested at the required concentration in 0.1% DMSO. Control wells contained 0.1% DMSO only and a maximum response was determined using a 10 xcexcM treatment of a known active compound. After a preincubation period of 40 minutes at 37xc2x0 C. in 5% CO2, cells were pulsed with sodium pyruvate to a final concentration of 0.5 mM (containing 1-14C sodium pyruvate (Amersham product CFA85) 0.18 Ci/mmole) for 12 minutes. The medium was then removed and transferred to a tube which was immediately sealed with a bung containing a suspended centre well. Absorbent within the centre well was saturated with 50% phenylethylamine, and CO2 in the medium released by the addition of 0.2 xcexcl 60% (w/v) perchloric acid (PCA). Released 14CO2 trapped in the absorbent was determined by liquid scintillation counting. The ED50 for a test compound was determined in the usual way using results from 7 concentrations of the compound.
(c) In vivo Elevation of PDH Activity
The capacity of compounds to increase the activity of PDH in relevant tissues of rats may be measured using the test described hereinafter. Typically an increase in the proportion of PDH in its active, nonphosphorylated form may be detected in muscle, heart, liver and adipose tissue after a single administration of an active compound. This may be expected to lead to a decrease in blood glucose after repeated administration of the compound. For example a single administration of DCA, a compound known to activate PDH by inhibition of PDH kinase (Whitehouse, Cooper and Randle (1974) Biochem. J. 141, 761-774) 150 mg/kg, intraperitoneally, increased the proportion of PDH in its active form (Vary et al. (1988) Circ. Shock 24, 3-18) and after repeated administration resulted in a significant decrease in plasma glucose (Evans and Stacpoole (1982) Biochem. Pharmacol.31, 1295-1300).
Groups of rats (weight range 140-180 g) are treated with a single dose or multiple doses of the compound of interest by oral gavage in an appropriate vehicle. A control group of rats is treated with vehicle only. At a fixed time after the final administration of compound, animals are terminally anaesthetised, tissues are removed and frozen in liquid nitrogen. For determination of PDH activity, muscle samples are disrupted under liquid nitrogen prior to homogenisation by one thirty-second burst in a Polytron homogenizer in 4 volumes of a buffer containing 40 mM potassium phosphate pH 7.0, 5 mM EDTA, 2 mM DTT, 1% Triton X-100, 10 mM sodium pyruvate, 10 xcexcM phenylmethylsulphonyl chloride (PMSF) and 21 xcexcg/ml each of leupeptin, pepstain A and aprotinin. Extracts are centrifuged before assay. A portion of the extract is treated with PDH phosphatase prepared from pig hearts by the method of Siess and Wieland (Eur. J. Biochem (1972) 26, 96): 20 xcexcl extract, 40 xcexcl phosphatase (1:20 dilution), in a final volume of 125 xcexcl containing 25 mM magnesium chloride, 1 mM calcium chloride. The activity of the untreated sample is compared with the activity of the dephosphorylated extract thus prepared. PDH activity is assayed by the method of Stansbie et al., (Biochem. J. (1976) 154, 225). 50 xcexcl Extract is incubated with 0.75 mM NAD, 0.2 mM CoA, 1.5 mM thiamine pyrophosphate (TPP) and 1.5 mM sodium pyruvate in the presence of 20 xcexcg/ml p-(p-amino-phenylazo) benzene sulphonic acid (AABS) and 50 mU/ml arylamine transferase (AAT) in a buffer containing 100 mM tris(hydroxymethyl)aminomethane, 0.5 mM EDTA, 50 mM sodium fluoride, 5 mM 2-mercaptoethanol and 1 mM magnesium chloride pH 7.8. AAT is prepared from pigeon livers by the method of Tabor et al. (J. Biol. Chem. (1953) 204, 127). The rate of acetyl CoA formation is determined by the rate of reduction of AABS which is indicated by a decrease in optical density at 460 nm.
Liver samples are prepared by an essentially similar method, except that sodium pyruvate is excluded from the extraction buffer and added to the phosphatase incubation to a final concentration of 5 mM.
Treatment of an animal with an active compound results in an increase in the activity of PDH complex in tissues. This is indicated by an increase in the amount of active PDH (determined by the activity of untreated extract as a percentage of the total PDH activity in the same extract after treatment with phosphatase).
According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula (I) as defined hereinbefore or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I) or a pharmaceutically acceptable salt of said compound or said ester, in association with a pharmaceutically acceptable excipient or carrier.
According to an additional aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula (Ie) as defined hereinbefore or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (Ie) or a pharmaceutically acceptable salt of said compound or said ester, in association with a pharmaceutically acceptable excipient or carrier.
According to an additional further aspect of the invention there is provided a pharmaceutical composition which comprises a compound selected from N-(4-benzoyl-2,6-dimethylphenyl)-2-hydroxy-2-methyl-3,3,3-trifluoropropanamide or 3-hydroxy-3-trifluoromethyl-1-(2-chloro-5-trifluoromethylphenyl)-4,4,4-trifluorobut-1-yne or a pharmaceutically acceptable in vivo cleavable ester of said compound or a pharmaceutically acceptable salt of said compound or said ester, in association with a pharmaceutically acceptable excipient or carrier.
The composition may be in a form suitable for oral administration, for example as a tablet or capsule, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) for example as a sterile solution, suspension or emulsion, for topical administration for example as an ointment or cream or for rectal administration for example as a suppository. In general the above compositions may be prepared in a conventional manner using conventional excipients.
The compositions of the present invention are advantageously presented in unit dosage form. The compound will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg. A unit dose in the range, for example, 1-100 mg/kg, preferably 1-50 mg/kg is envisaged and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient.
According to a further aspect of the present invention there is provided a compound of the formula (I) or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I) or a pharmaceutically acceptable salt of said compound or said ester as defined hereinbefore for use in a method of treatment of the human or animal body by therapy.
We have found that compounds of the present invention elevate PDH activity and are therefore of interest for their blood glucose-lowering effects.
A further feature of the present invention is a compound of formula (I) or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I) or a pharmaceutically acceptable salt of said compound or said ester for use as a medicament, conveniently a compound of formula (I) or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I);
or a pharmaceutically acceptable salt of said compound or said ester for use as a medicament for producing an elevation of PDH activity in a warm-blooded animal such as a human being.
Thus according to a further aspect of the invention there is provided the use of a compound of the formula (I) or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I) or a pharmaceutically acceptable salt of said compound or said ester in the manufacture of a medicament for use in the production of an elevation of PDH activity in a warm-blooded animal such as a human being.
According to a further feature of the invention there is provided a method for producing an elevation of PDH activity in a warm-blooded animal, such as a human: being, in need of such treatment which comprises administering to said animal an effective amount of a compound of formula (a) or a pharmaceutically acceptable in vivo cleavable ester of said compound of formula (I) or a pharmaceutically acceptable salt of said compound or said ester as defined hereinbefore.
As stated above the size of the dose required for the therapeutic or prophylactic treatment of a particular disease state will necessarily be varied depending on the host treated, the route of administration and the severity of the illness being treated. Preferably a daily dose in the range of 1-50 mg/kg is employed. However the daily dose will necessarily be varied depending upon the host treated, the particular route of administration, and the severity of the illness being treated. Accordingly the optimum dosage may be determined by the practitioner who is treating any particular patient.
The elevation of PDH activity described herein may be applied as a sole therapy or may involve, in addition to the subject of the present invention, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. For example in the treatment of diabetes mellitus chemotherapy may include the following main categories of treatment:
i) insulin;
ii) insulin secretagogue agents designed to stimulate insulin secretion (for example glibenclamide, tolbutamide, other sulphonylureas);
iii) oral hypoglycaemic agents such as metformin, thiazolidinediones;
iv) agents designed to reduce the absorption of glucose from the intestine (for example acarbose);
v) agents designed to treat complications of prolonged hyperglycaemia;
vi) other agents used to treat lactic acidaemia;
vii) inhibitors of fatty acid oxidation;
viii) lipid lowering agents;
ix) agents used to treat coronary heart disease and peripheral vascular disease such as aspirin, pentoxifylline, cilostazol; and/or
x) thiamine.
As stated above the compounds defined in the present invention are of interest for their ability to elevate the activity of PDH. Such compounds of the invention may therefore be useful in a range of disease states including diabetes mellitus, peripheral vascular disease, (including intermittent claudication), cardiac failure and certain cardiac myopathies, myocardial ischaemia, cerebral ischaemia and reperfusion, muscle weakness, hyperlipidaemias, Alzheimers Disease and/or atherosclerosis.
In addition to their use in therapeutic medicine, the compounds of formula (I) and their pharmaceutically acceptable salts are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of elevators of PDH activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
It is to be understood that where the term xe2x80x9cetherxe2x80x9d is used anywhere in this specification it refers to diethyl ether.